High specificity and sensitivity immunosorbent diagnostic assays with simultaneous resolution of multiple antibody isotypes

Abstract

Compositions and methods are provided for diagnosis of infections. The patterns of antibody isotype, subtype and glycosylation provide for a signature pattern that can identify infective agents and patient response to infection. Patients likely to benefit from therapeutic intervention can be discriminated from patients that have a low probability of responsiveness. Therapies are also provided.

What is claimed is: 
     
         1 . A method of characterizing an immune response to a pathogen by an individual, the method comprising:
 collecting at least one antibody-containing sample from the individual;   contacting said at least one antibody-containing sample from the individual with a diagnostic pathogen;   contacting the diagnostic pathogen with one or more isotype-specific or glycosylation-specific reagents, which reagents are operably linked to a detectable moiety; and   analyzing the diagnostic pathogen for the presence of bound isotype-specific or glycosylation-specific reagents to determine the presence and type of pathogen-specific antibodies, wherein the presence and type is indicative of a pathogen infection and immune response.   
     
     
         2 . The method of  claim 1 , wherein the diagnostic pathogen is an intact pathogen, genetically modified to express a fluorophore. 
     
     
         3 . The method of  claim 2 , wherein the fluorophore is a fluorescent protein. 
     
     
         4 . The method of  claim 3 , wherein the fluorescent protein is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), and analogs thereof. 
     
     
         5 . The method of any of  claims 1 - 4 , wherein the diagnostic pathogen is a live pathogen or a fixed pathogen. 
     
     
         6 . The method of any of  claims 1 - 5 , wherein the diagnostic pathogen is a clinical isolate or an environmental isolate. 
     
     
         7 . The method of any of  claims 1 - 6 , wherein the diagnostic pathogen is from a cell line or cell culture. 
     
     
         8 . The method of any of  claims 1 - 7 , wherein the diagnostic pathogen is further genetically modified to eliminate expression of proteins and other epitopes that are highly conserved among pathogens. 
     
     
         9 . The method of  claim 8 , wherein the epitopes are present on cell surface proteins. 
     
     
         10 . The method of  claim 9 , wherein the cell surface proteins are flagellar proteins. 
     
     
         11 . The method of  claim 10 , wherein the flagellar proteins are one or both of fliH and flil proteins of  Borrelia.    
     
     
         12 . The method of any of  claims 1 - 11 , wherein the pathogen is a cellular pathogen. 
     
     
         13 . The method of  claim 12 , wherein the pathogen is selected from a bacterium, a fungus, and a protozoan. 
     
     
         14 . The method of  claim 13 , wherein the bacterium is a Spirochaetes. 
     
     
         15 . The method of  claim 14 , wherein the Spirochaetes is a  Borrelia  sp. 
     
     
         16 . The method of any of  claims 1 - 15  wherein the infectious pathogen is a tick-borne pathogen. 
     
     
         17 . The method of  claim 16 , wherein the tick-borne pathogen is  Borrelia burgdorferi.    
     
     
         18 . The method of any of  claims 1 - 17 , wherein the isotype specific reagent is an antibody that recognizes one of IgM, IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgA, IgA1, IgA2, IgE, IgD or Ig specific glycosylation sites (such as IgA1-G2S1 or IgG4-G0F). 
     
     
         19 . The method of  claim 18 , wherein a cocktail of 2 or more uniquely-labeled isotype/subtype/glycosylation-specific reagents is contacted with the sample. 
     
     
         20 . The method of any of  claims 1 - 19 , wherein analysis is performed by a method selected from flow cytometry, mass cytometry, sequencing or PCR of sequence barcoded antibodies, and high dimensional/multi-parameter microscopy. 
     
     
         21 . The method of any of  claims 1 - 20  wherein the pathogen is a vaccine strain and the presence and type of pathogen-specific antibodies is indicative of response to vaccine immunization.
 An additional claim on bead or chip based protein or peptide arrays as the “pathogen” 
 
     
     
         22 . The method of any of  claims 1 - 21 , further comprising treating the individual in accordance with the analysis, optionally with one or more of an antihistamine, anti-IgE agent or mast cell stabilizing agent where pathogen-specific IgE antibodies are present. 
     
     
         23 . The method of any of  claims 1 - 22 , wherein the antibody-containing sample is a blood sample. 
     
     
         24 . The method of any of  claims 1 - 23 , further comprising monitoring the immune response to the pathogen by the individual for a period of time by repeating a)-d) at a plurality of time points. 
     
     
         25 . The method of  claim 24 , wherein a first antibody-containing sample is collected from the individual at a first time point and a second antibody-containing sample is collected from the individual at a later second time point, wherein detection of increased levels of one or more pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more pathogen-specific antibodies in the first antibody-containing sample indicates that the infection by the pathogen is worsening, and decreased levels of the one or more pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more pathogen-specific antibodies in the first antibody-containing sample indicates that the infection by the pathogen is improving. 
     
     
         26 . The method of  claim 25 , further comprising monitoring the efficacy of a therapy for treating the infection by the pathogen, wherein the first antibody-containing sample is collected from the individual before the patient undergoes the therapy and the second antibody-containing sample is collected from the individual after the patient undergoes the therapy, wherein detection of increased levels of the one or more pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more pathogen-specific antibodies in the first antibody-containing sample indicates that the infection by the pathogen is worsening or not responding to the therapy, and decreased levels of the one or more pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more pathogen-specific antibodies in the first antibody-containing sample indicates that the infection by the pathogen is improving. 
     
     
         27 . A diagnostic pathogen for use in the methods of any of  claims 1 - 26 . 
     
     
         28 . A method of diagnosing and treating an individual with Lyme disease, the method comprising:
 collecting at least one antibody-containing sample from the individual;   contacting said at least one antibody-containing sample from the individual with a diagnostic bait displaying a plurality of  Borrelia burgdorferi  pathogen antigens;   contacting the diagnostic bait with one or more isotype-specific or glycosylation-specific reagents, which reagents are operably linked to a detectable moiety;   analyzing the diagnostic bait for the presence of bound isotype-specific or glycosylation-specific reagents to determine the presence and type of  Borrelia burgdorferi  pathogen-specific antibodies, wherein the presence and type is indicative of a  Borrelia burgdorferi  infection and an immune response to the  Borrelia burgdorferi  pathogen;   diagnosing the individual with Lyme disease if the presence of one or more  Borrelia burgdorferi  pathogen-specific antibodies is detected, and   treating the individual for Lyme disease if the presence of one or more  Borrelia burgdorferi  pathogen-specific antibodies is detected, wherein one or more of an antihistamine; anti-IgE agent or mast cell stabilizing agent is administered to the individual if the presence of  Borrelia burgdorferi  pathogen-specific immunoglobulin E (IgE) antibodies is detected.   
     
     
         29 . The method of  claim 28 , wherein the antihistamine inhibits a histamine receptor selected from the group consisting of H1, H2, H3, and H4. 
     
     
         30 . The method of  claim 28 , wherein the antihistamine is selected from the group consisting of cimetidine, ranitidine, Benadryl, diphenhydramine, loratadine, doxepin, thioperamide, and clobenpropit. 
     
     
         31 . The method of any of  claims 28 - 30 , further comprising depleting or stabilizing mast cells in the individual if the presence of  Borrelia burgdorferi  pathogen-specific immunoglobulin E (IgE) antibodies is detected. 
     
     
         32 . The method of  claim 31 , wherein said mast cells are depleted by administering anti-c-kit therapy. I think we should have a list of possible mast cell stabilizing drugs as well and/or refer to the tables in that review 
     
     
         33 . The method of  claim 32 , further comprising administering anti-CD47 therapy. 
     
     
         34 . The method of any of  claims 28 - 33 , further comprising depleting IgE producing B cells in the individual if the presence of  Borrelia burgdorferi  pathogen-specific immunoglobulin E (IgE) antibodies is detected. 
     
     
         35 . The method of any of  claims 28 - 34 , wherein anti-IgE therapy comprises IgE blockade or linkage of IgE specific antibodies to a different isotype with beneficial effector functions. 
     
     
         36 . The method of  claim 35 , wherein the isotype is IgG. 
     
     
         37 . The method of any of  claims 28 - 36 , further comprising dampening an IgE response by cytokine blockade of one or more cytokines selected from the group consisting of IL-4, IL-5, and IL-13. 
     
     
         38 . The method of any of  claims 28 - 37 , wherein the diagnostic bait s a diagnostic  Borrelia burgdorferi  pathogen. 
     
     
         39 . The method of  claim 38 , wherein the diagnostic  Borrelia burgdorferi  pathogen is genetically modified to express a fluorophore. 
     
     
         40 . The method of  claim 39 , wherein the fluorophore is a fluorescent protein. 
     
     
         41 . The method of  claim 40  wherein the fluorescent protein is selected from green fluorescent protein (GFP), red fluorescent protein (RFP), and analogs thereof. 
     
     
         42 . The method of any of  claims 38 - 41 , wherein the diagnostic  Borrelia burgdorferi  pathogen is a  Borrelia burgdorferi  clinical isolate or an environmental isolate. 
     
     
         43 . The method of any of  claims 38 - 41 , wherein the diagnostic  Borrelia burgdorferi  pathogen is from a  Borrelia burgdorferi  cell line or cell culture. 
     
     
         44 . The method of any of  claims 38 - 43 , wherein the diagnostic pathogen is further genetically modified to eliminate expression of proteins and other epitopes that are highly conserved among pathogens. 
     
     
         45 . The method of  claim 44 , wherein the epitopes are present on cell surface proteins. 
     
     
         46 . The method of  claim 45 , wherein the cell surface proteins are fliH or flil proteins of  Borrelia  or a combination thereof. 
     
     
         47 . The method of any of  claims 38 - 46 , wherein the diagnostic pathogen is a live pathogen or a fixed pathogen. 
     
     
         48 . The method of any of  claims 28 - 47 , wherein the diagnostic bait is an antigen array comprising  Borrelia burgdorferi  pathogen proteins or peptide epitopes. 
     
     
         49 . The method of any of  claims 28 - 48 , wherein the isotype specific reagent is an antibody that recognizes one of IgM, IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgA, IgA1; IgA2, IgE, IgD or Ig specific glycosylation sites (such as IgA1-G2S1 or IgG4-G0F). 
     
     
         50 . The method of any of  claims 28 - 49 , wherein analysis is performed by a method selected from flow cytometry, mass cytometry, sequencing or PCR of sequence barcoded antibodies, and high dimensional/multi-parameter microscopy. 
     
     
         51 . The method of any of  claims 28 - 50 , further comprising monitoring the immune response to the  Borrelia burgdorferi  pathogen by the individual for a period of time by repeating at a plurality of time points. 
     
     
         52 . The method of  claim 51 , wherein a first antibody-containing sample is collected from the individual at a first time point and a second antibody-containing sample is collected from the individual at a later second time point, wherein detection of increased levels of one or more  Borrelia burgdorferi  pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more  Borrelia burgdorferi  pathogen-specific antibodies in the first antibody-containing sample indicates that the Lyme disease is worsening, and decreased levels of the one or more  Borrelia burgdorferi  pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more  Borrelia burgdorferi  pathogen-specific antibodies in the first antibody-containing sample indicates that the Lyme disease is improving. 
     
     
         53 . The method of  claim 52 , further comprising monitoring the efficacy of a therapy for treating Lyme disease, wherein the first antibody-containing sample is collected from the individual before the patient undergoes the therapy and the second antibody-containing sample is collected from the individual after the patient undergoes the therapy, wherein detection of increased levels of the one or more  Borrelia burgdorferi  pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more  Borrelia burgdorferi  pathogen-specific antibodies in the first antibody-containing sample indicates that the Lyme disease is worsening or not responding to the therapy, and decreased levels of the one or more  Borrelia burgdorferi  pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more  Borrelia burgdorferi  pathogen-specific antibodies in the first antibody-containing sample indicates that the Lyme disease is improving. 
     
     
         54 . The method of any of  claims 28 - 53 , wherein said treating the individual for Lyme disease further comprises administering an antibiotic. 
     
     
         55 . A  Borrelia burgdorferi  diagnostic pathogen for use in the methods of any of  claims 28 - 47  and  49 - 54 . 
     
     
         56 . A kit comprising the  Borrelia burgdorferi  diagnostic pathogen of  claim 55  and one or more isotype-specific or glycosylation-specific reagents for detecting  Borrelia burgdorferi  pathogen-specific antibodies. 
     
     
         57 . The kit of  claim 56 , wherein the isotype-specific reagents comprise IgE-specific reagents for detecting  Borrelia burgdorferi  pathogen-specific IgE antibodies. 
     
     
         58 . The kit of  claim 56  or  57 , further comprising an antihistamine or an anti-IgE therapeutic agent. 
     
     
         59 . The kit of  claim 58 , wherein the antihistamine is selected from the group consisting of cimetidine, ranitidine, Benadryl, diphenhydramine, loratadine, doxepin, thioperamide, and clobenpropit.